Rop protein

Rop (Repressor Of Primer) is a small homodimeric RNA-binding protein that is involved in the regulation of copy number of the ColE1 plasmids of E.coli, where it is encoded. Its structure has been studied using both X-ray crystallography (Banner et al., 1987) and NMR (Eberle et al., 1991). Each monomer has molecular weight of about 7.500 Da and it consists of 63 amino acids that form two α-helices connected by  a loop of four amino acids (L29, D30, A31, D32). The two monomers are related with a 2-fold symmetry axis.

4-α-helical bundle
Rop is the paradigm of a canonical 4-α-helical bundle and its apparent structural simplicity of its folding rendered it as a model system for investigating the sequence-structure relationships in the folding and dynamics of 4-α-helix. The four α-helices are amphipathic, packed in an antiparallel fashion and display a specific pattern of hydrophobic and hydrophilic amino acids, of the type (a,b,c,d,e,f,g)n, which is repeated every seven residues (heptad pattern). Positions a and d are generally hydrophobic and the side chains of these residues are packed in the central part of the structure according to the “knobs in holes” model forming the hydrophobic core. The heptad periodicity in the sequence of Rop is disrupted only once and leads to the formation of the loop. Ala31 has a crucial role in the formation of the loop region as it is the only amino acid that simultaneously forms hydrogen bond to both helices.

Mutants
Numerous mutations in the loop region of Rop have been produced:
 * deletion of 5 a/a of loop (1qx8)
 * site-directed mutants in loop
 * replacement and insertion of glycine residues in loop
 * restored of heptad pattern in loop region) (1nkd)
 * a single alanine to proline substitution (1b6q) (Pro31, unlike Ala31, is more confor-mationally constrained, the dihedral angles of Ala31 in the wild type molecule are prohibited to Pro, and leads to a folding pathway for a thermodynamically less stable conformation.)
 * re-engineering topology of the homodimeric ROP protein into a single-chain 4-helix bundle(1yo7)
 * ALA2ILE2-6, repacted the hydrophobic core and a new fold (1f4n)

Additional Resources
For additional information, see: DNA Replication, Repair, and Recombination